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Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-β1 (TGF-β1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-β1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis., Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality., Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance., Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mehta, Sanz-Magallón Duque de Estrada, Denneny, Foster, Turner, Mayer, Milighetti, Platé, Worlock, Yoshida, Brown, Nikolić, Chain, Noursadeghi, Chambers, Porter and Tomlinson.)

Fibrosis is the concluding pathological outcome and major cause of morbidity and mortality in a number of common chronic inflammatory, immune-mediated and metabolic diseases. The progressive deposition of a collagen-rich extracellular matrix (ECM) represents the cornerstone of the fibrotic response and culminates in organ failure and premature death. Idiopathic pulmonary fibrosis (IPF) represents the most rapidly progressive and lethal of all fibrotic diseases with a dismal median survival of 3.5 years from diagnosis. Although the approval of the antifibrotic agents, pirfenidone and nintedanib, for the treatment of IPF signalled a watershed moment for the development of anti-fibrotic therapeutics, these agents slow but do not halt disease progression or improve quality of life. There therefore remains a pressing need for the development of effective therapeutic strategies. In this article, we review emerging therapeutic strategies for IPF as well as the pre-clinical and translational approaches that will underpin a greater understanding of the key pathomechanisms involved in order to transform the way we diagnose and treat pulmonary fibrosis., Competing Interests: Declaration of competing interest RCC declares being in receipt of research funding in the form of a research collaboration agreement with Chiesi Farmaceutici outside the submitted work., (Copyright © 2023. Published by Elsevier Ltd.)

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted Dl CO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.

Introduction: The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD)., Methods and Analysis: The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment., Ethics and Dissemination: All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals., Conclusion: This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Introduction: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection., Methods: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene -based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression., Results: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor β1 (TGF-β1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-β1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts., Conclusion: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions., Competing Interests: Competing interests: RCC received grants from GSK via her institution during the conduct of the study; consultancy fees from Pieris Pharmaceuticals, consultancy fees from Chiesi, consultancy fees from Theravance Biopharma outside the submitted work; and RCC spouse was an employee of GSK during the time this work was conducted. This potential COI was managed by a research framework agreement between UCL and GSK. DG and PFM received salary from this research framework agreement. RM, AT, LME and AB were employees of GlaxoSmithKline during the time this work was performed and hold GSK shares. AJF has received a research grant from GSK to support work contained in this manuscript. RH and GM were employees of Epistem with a commercial interest in the provision of services in the area of investigation. TMM has, via his institution, received industry-academic funding from GSK, R&D and UCB and has received consultancy or speakers fees from Apellis, AstraZeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, ProMetic, Respivnat, Roche, Samumed and UCB. MJH reports non-financial support from Achilles Therapeutics outside the submitted work. MP, RJM and REH have nothing to disclose., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Idiopathic pulmonary fibrosis (IPF) is characterised by the progressive deposition of excessive extracellular matrix proteins within the lung parenchyma and represents the most rapidly progressive and fatal of all fibrotic conditions. Current anti-fibrotic drugs approved for the treatment of IPF fail to halt disease progression and have significant side-effect profiles. Therefore, there remains a pressing need to develop novel therapeutic strategies for IPF. Mammalian target of rapamycin (mTOR) forms the catalytic subunit of two complexes, mTORC1 and mTORC2. mTORC1 acts as critical cellular sensor which integrates intracellular and extracellular signals to reciprocally regulate a variety of anabolic and catabolic processes. The emerging evidence for a critical role for mTORC1 in influencing extracellular matrix production, metabolism, autophagy and senescence in the setting of IPF highlights this axis as a novel therapeutic target with the potential to impact multiple IPF pathomechanisms., Competing Interests: Conflict of interest: M. Platé has nothing to disclose. Conflict of interest: D. Guillotin reports other funding from GlaxoSmithKline, during the conduct of the study. Conflict of interest: R. Chambers reports grants from GlaxoSmithKline during the conduct of the study., (Copyright ©ERS 2020.)

The #ERSCongress is the largest respiratory conference worldwide. This article points out why 
@ERSTalk Early Career Members can especially benefit from attending this huge event and gives upcoming fellowship deadlines. http://ow.ly/3GQ530o8Ezh., Competing Interests: Conflict of interest: N. Ubags has nothing to disclose. Conflict of interest: M. Platé has nothing to disclose. Conflict of interest: I. Almendros has nothing to disclose. Conflict of interest: S. Bartel reports a grant and personal fees for advisory board membership from Bencard Allergie GmbH outside the submitted work.

The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-β 1 (TGF-β 1 ), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-β 1 -induced ATF4 production depended on cooperation between canonical TGF-β 1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-β 1 -mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myofibroblasts are the key effector cells responsible for excessive extracellular matrix deposition in multiple fibrotic conditions, including idiopathic pulmonary fibrosis (IPF). The PI3K/Akt/mTOR axis has been implicated in fibrosis, with pan-PI3K/mTOR inhibition currently under clinical evaluation in IPF. Here we demonstrate that rapamycin-insensitive mTORC1 signaling via 4E-BP1 is a critical pathway for TGF-β 1 stimulated collagen synthesis in human lung fibroblasts, whereas canonical PI3K/Akt signaling is not required. The importance of mTORC1 signaling was confirmed by CRISPR-Cas9 gene editing in normal and IPF fibroblasts, as well as in lung cancer-associated fibroblasts, dermal fibroblasts and hepatic stellate cells. The inhibitory effect of ATP-competitive mTOR inhibition extended to other matrisome proteins implicated in the development of fibrosis and human disease relevance was demonstrated in live precision-cut IPF lung slices. Our data demonstrate that the mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis with potential implications for the development of novel anti-fibrotic strategies.

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3-8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer.

Rationale: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose., Methods: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung., Results: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF., Conclusions: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway., Trial Registration Number: NCT01725139, pre-clinical., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) is a versatile nanocomposite biomaterial with growing applications as a bioscaffold for tissue engineering. Integration of synthetic implants with host tissue can be problematic but could be improved by topographical modifications. We describe optimization of POSS-PCU by dispersion of porogens (sodium bicarbonate (NaHCO3), sodium chloride (NaCl) and sucrose) onto the material surface, with the principle aim of increasing surface porosity, thus providing additional opportunities for improved cellular and vascular ingrowth. We assess the effect of the porogens on the material's mechanical strength, surface chemistry, wettability and cytocompatibilty. Surface porosity was characterized by scanning electron microscopy (SEM). There was no alteration in surface chemistry and wettability and only modest changes in mechanical properties were detected. The size of porogens correlated well with the porosity of the construct produced and larger porogens improved interconnectivity of spaces within constructs. Using primary human bronchial epithelial cells (HBECs) we demonstrate moderate in vitro cytocompatibility for all surface modifications; however, larger pores resulted in cellular aggregation. These cells were able to differentiate on POSS-PCU scaffolds. Implantation of the scaffold in vivo demonstrated that larger pore sizes favor cellular integration and vascular ingrowth. These experiments demonstrate that surface modification with large porogens can improve POSS-PCU nanocomposite scaffold integration and suggest the need to strike a balance between the non-porous surfaces required for epithelial coverage and the porous structure required for integration and vascularization of synthetic scaffolds in future construct design., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes. In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Proteinase-activated receptor-1 (PAR-1) plays a key role in mediating the interplay between coagulation and inflammation in response to injury. The aim of this study was to investigate the role of the promoter single-nucleotide polymorphism (SNP) rs2227744G>A in modulating PAR-1/F2R gene expression in the context of chronic obstructive pulmonary disease (COPD) and COPD exacerbations. The function of the rs2227744G>A SNP was investigated by using reporter gene assays. The frequency of the polymorphism in the UK population was assessed by genotyping 8,579 healthy individuals from the Whitehall II and English Longitudinal Study of Ageing cohorts. The rs2227744G>A SNP was genotyped in a carefully phenotyped cohort of 203 COPD cases and matched controls. The results were further replicated in two different COPD cohorts. The minor allele of the rs2227744G>A polymorphism was found to increase F2R expression by 2.6-fold (P < 0.001). The rs2227744G>A SNP was not significantly associated with COPD, or with lung function, in all cohorts. The minor allele of the SNP was found to be associated with protection from frequent exacerbations (P = 0.04) in the cohort of COPD patients for which exacerbation frequency was available. Considering exacerbations as a continuous variable, the presence of the minor allele was associated with a significantly lower COPD exacerbation rate (3.03 vs. 1.98 exacerbations/year, Mann-Whitney U-test P = 0.04). Taken together, these data do not support a role for the rs2227744G>A F2R polymorphism in the development of COPD but suggest a protective role for this polymorphism from frequent exacerbations. Studies in separate cohorts to replicate these findings are warranted.

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB. In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.

The high-T(c) superconductor Tl(2)Ba(2)CuO(6 + delta) is studied by angle-resolved photoemission spectroscopy. For a very overdoped T(c) = 30 K sample, the Fermi surface consists of a single large hole pocket centered at (pi, pi) and is approaching a topological transition. Although a superconducting gap with d(x(2)-y(2)) symmetry is tentatively identified, the quasiparticle evolution with momentum and binding energy exhibits a marked departure from the behavior observed in under and optimally doped cuprates. The relevance of these findings to scattering, many-body, and quantum-critical phenomena is discussed.

Of 50 B. fragilis strains isolated from clinical samples we have demonstrated that 24 (48%) possess an adhesin that mediates a neuraminidase-dependent attachment of B. fragilis to mammalian epithelial cells, but does not mediate any association with human polymorphonuclear leucocytes. This ligand interacts with a mammalian cell receptor that contains a galactoside residue, exposed after neuraminidase pretreatment. Our results suggest a possible role for cell associated neuraminidase in mediating a two step adherence mechanism.